The striking genetic heterogeneity of the RNA genome of HCV is well recognized. On the basis of molecular relatedness, HCV is classified into 11 major genotypes: 1 through 11, among which first six are major player of infection globally. On the basis of phylogenetic analysis, over 80 subtypes and minor variants referred to as “quasispecies are existing, which differ by 20% to 23% on the basis of full length genomic sequence comparisons subtype. Identification of HCV genotype does not influence disease presentation but is important for its predictive value in term of antiviral therapy, counseling and management. Counseling is indeed a necessity in order to minimize the risk of transmission of HCV infection to others. This study investigated the subtypes of HCV infection and correlate genotypes of the HD patients with the demographic data and risk factors. This study also evaluation the prevalence of HCV with the past studies in HD patients conducting in different regions of Pakistan.
Study Sample and Data Collection
384 HD patients were randomly selected collected from three hospitals of Peshawar, Khyber Pakhtunkhwa: Khyber Teaching Hospital, National Diagnostic Dialysis Center and Dialysis Ward Hayatabad Medical Complex. All patients were briefed about the study and proper willing consent was signed.
All patients were interviewed for demographic data and risk factors to HCV infections including history of number of blood transfusion, intravenous drug use (IDU), surgical interventions, and dental treatment, multiple sexual partners, barber shop, piercing instruments and exposure to known HCV-positive persons, number of years on dialysis and change of the center. Blood (5CC in sterile syringes) were collected from HD patients; sera were separated in two aliquots and frozen at -70°C for HCV RNA detection and genotyping.
Antibodies Screening
All the subjects were screened for anti HCV antibodies third generation test according to the manufacture instructions (Accurate Diagnostics USA).
RNA Isolation, PCR amplification and Detection
RNA was isolated from 100 μL serum, using RNAzole RNA purification kit (Trizole Inc. USA) as per manufacturer protocols. HCV RNA was Reverse transcribed with 200 U of Maloney murine leukemia virus reverse transcriptase (Fermentas USA) and amplified with nested primers specific for 5′ untranslated region of HCV genome. Amplified product was subjected in 2% agarose gel for electrophoresis.
HCV genotyping
HCV RNA positive samples were genotyped with primers specific for core region of different HCV types by type specific PCR as mentioned elsewhere.
For contamination control RNA extraction, cDNA amplification and electrophoresis were carried in separate areas. Both negative and positive controls were run parallel to the patient samples in each batch.